Method for using division arrested cells in screening assays

ABSTRACT

Division arrested cells are used in screening assays to determine the effect of a substance of interest on the cells. The division arrested cells can be used in drug screening assays, signal transduction assays, and are especially useful in large scale, high throughput assays.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.11/363,983, filed Feb. 27, 2006, which is a divisional of U.S. patentapplication Ser. No. 10/251,467, filed Sep. 20, 2002, now U.S. Pat. No.7,045,281, which applications are entirely incorporated herein byreference.

FIELD OF THE INVENTION

This invention relates to methods for screening for substances ofinterest. More particularly, it relates to screening assays whichutilize cells in a division arrested state which, nonetheless, functioneffectively in assays where dividing cells would normally be used. Theadvantages of the system will be seen in the disclosure which follows.

BACKGROUND AND PRIOR ART

Cell based screening assays are tools well known to biologists. In theassays, one investigates compounds of interest to determine, e.g. if thecompounds modulate one or more biological processes of interest.

Among the cell based systems which are used are those which measurereporter activity, calcium activity assay, and so forth. These, and allother cell based assays are encompassed by the invention.

One area where cell based screening assays have become widely acceptedis the high throughput analysis of materials for use as pharmaceuticals.These assays are useful and desirable because compounds which areidentified initially in biochemical assays have been known to fail asdrug candidates later in the development process. The reasons for thisare many. In some cases, the compound does not permeate the cellreadily. In others, target binding capability is not predictive of thetarget modulating function, a feature that is, ultimately, a requirementof drug functions. Cell based screening assays are useful in that theyaddress a number of problems associated with animal model testing (e.g.,high expense, intensive labor, long assay period). High throughput cellbased screening assays can be scaled up via technologies such as“FLIPR,” “Leedseeker,” “VIPR,” and fluorescent, high speed cell-imaging.

In addition to assays such as those discussed supra, other commonlyused, cell based assays involve enzyme-reporter systems, cell activityassays with a fluorescent or calorimetric readout, and so forth. Anexample of such an assay is a Ca²⁺ mobilization assay to measureG-protein coupled receptor activity with the dye “Fluo-4.”

In addition to the advantages set forth supra, cell based assays have adistinct advantage in that they permit the user to determine thefunctional outcome, of the use of compounds. Properly designed assaysalso permit the artisan to select against the toxic compounds, whenscreening for active ones.

Carrying out high throughput, cell based assays present uniquechallenges to users. Unlike pure biochemical reagent like enzymes,proteins, and membrane receptor preparations, cells are live dynamicentities. Preparation and cultivation have to be tied to the actualscreening process.

Actively managed cell culture cycles have a recovery phase, when theyare split from near confluent cultures, followed by an early log growthphase, then a mid log, and a late log phase, leading to a stationaryphase if the culture is allowed to become confluent. Variances of thecellular processes and protein components at these different stages ofgrowth and replication occur constantly during these cell cultures ascells cycle through mitosis. These variances must be expected to affectbiological assays, and be a factor in the common phenomenon ofvariability in high throughput assays. One, but by no means the onlyexample of this, relates to the length of time over which assays arerun. There is generally an 8-36 hour period following the seeding ofcells during which the assay is run. The cells in the particular culturego through different phases of their culture cycle during this time, andit is not usual for the cells to be at the same point in the cycle atthe same time.

Further, the miniaturization of cell based screening assays isprogressing, with smaller and smaller numbers of cells being used. Asthis occurs, sensitivity of the assay to variability increases rapidlyand dramatically.

The critical factors of a good cell based screening assay are (i) a wellvalidated target, (ii) a sensitive readout, and (iii) extremely highconsistency of the cells that are used. The invention which is set forthin the disclosure which follows addresses this third issue. Theconsistent performance of the cells in an assay can be greatly affectedby changes in the level of target expression as a result of increasingcell passage number. In addition, a well characterized population ofcells can be division arrested, cryopreserved as a cell bank, and platedfor an assay without any additional passaging of the cells. In fact,division arrested cells may be plated and used over a period of up tofive (5) days without any further handling of the cells and without asignificant change in cell based responses.

Division arrested cells have been used in the art. Exemplary of this areCho, et al., Biochem. Mol. Bio. Int. 42:949 56 (1997); Yao, et al., Mol.Pharmacol 57:422 30 (1997); Fueger, et al., J. Nucl. Med. 42:1856 62(2001); Muller, et al., J. Exp. Med. 188: 2033 45 (1998), and Kharbanda,et al., Nature 376 (6543):785-8 (1995). All of these references areincorporated by reference. Review of these, as well as other referenceswill show that these studies concern characterization of the cells,rather than their use in assays of the type described herein, such asdrug discovery, screening assays, and/or signal transduction assays,especially when these are carried out on a large scale, high throughputbasis as is required for industrial application.

These, as well as other features of the invention will be elaboratedupon in the disclosure which follows.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A 1C set forth FACS scans depicting secrotonin induction of Ca⁺⁺mobilization on division arrested cells, which are set forth in example1.

FIG. 2 shows the result of experiments, described in detail in example2. It shows carbachol induced, Ca²⁺ mobilization, observed via changesin fluorescent emission ratios of Fura2 loaded into cells that wereinduced.

FIG. 3 summarizes the result of experiments set forth in example 3,involving cells where division was arrested by irradiation, and whichwere treated with isoproterenol and reporter gene changes were measured.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS Example 1

These experiments describe screening assays designed to measure theinduction of Ca²⁺ mobilization by serotonin in growth arrested, NIH3T3cells.

NIH3T3 cells were stably transfected with cDNA encoding the knownreceptor “5HT2c” using standard methods. For information on thereceptor, see Julius, et al., Proc. Natl. Acad. Sci USA 87:928 32(1990), incorporated by reference. After the transfection, and apreliminary screen to make sure that the transfection was successful,NIH3T3 cells which expressed the (4500 mg/L), together with 1×penicillin-streptomycin solution and 10% (v/v) fetal bovine serum (FBS).This cell line is referred to as “NIH3T3-POP.”

In order to arrest the growth of the cells, they were exposed to 10μg/ml of mitomycin C, for 2.5 hours. Mitomycin C is well known for itsability to arrest cell growth by blocking microtubule mobility, therebyarresting cell division. The cells were frozen, 2.5 hours aftertreatment, using standard protocols.

As a control, cells which were not exposed to mitomycin C were frozenusing the same procedure. Samples of both treated and control cells werethawed, using standard methods, and plated for 24 hours, prior toharvest. Harvesting was accomplished by adding 7 ml of an enzyme free,cell dissociation solution, (e.g., EnzymeFree Cell DissociationSolution, Specialty Media, catalog number S-014) or with 0.5 mM EDTA, tocells. The number of cells used in each experiment was 4×10⁵ cells/ml orabout 4 4.5×10⁶ total cells. Such solutions are widely available, andare well known to the skilled artisan. This treatment dissociated cellsfrom the flask, and aggregates were then broken up by repeated pipettingof the suspensions, up and down in the flasks, so as to provide a goodproportion of single cells, as this is necessary for the FACS analysiswhich followed.

Cells were pelleted, and then resuspended in 6 ml of Indo-1 loadingbuffer.

Cells were loaded with Indo-1 AM dye by adding 2 mg/ml of Indo-1 AMstock, in DMSO, to the cell suspensions to a final concentration of 10μg/1.2 mls. Cells were exposed to Indo-1 AM for seven (7) minutes atroom temperature and then diluted up to a final volume of 10 mls withIndo-1 loading buffer and pelleted.

In order to measure and to analyze Ca²⁺ mobilization, a commerciallyavailable cell sorter was used. Excitation was set at 360 nm, andemissions were set at both 400 (+/−15 nm) and 500 (+/−20)nm. Theemissions were monitored simultaneously, and the emission ratio at 400nm/500 nm was used to report the intracellular rise of Ca²⁺concentration. Untreated cells were used to set a baseline ratio.

Cells were resuspended in a buffer and loaded into a syringe. Cells wereinjected continuously into the flow cytometer to be sampled and toprovide a baseline value. A substance such as an antagonist can be usedto pretreat the cells, with a second material, such as an agonist beinginjected continuously and simultaneously through, e.g., a secondsyringe, via a connecting means, such as a standard Y-connector. Cellsare then exposed to the test substance, analyzed, and changes frombaseline measured.

The data of the experiment are summarized in FIG. 1. Cells which hadbeen division arrested (POP Ar 10 μM 5HT panel in FIG. 1) responded in amanner similar to those which were not (POP Gr 10 μM 5HT panel in FIG.1). In brief, “% positive” was calculated by taking the percentage inthe positive region (i.e., cells demonstrating an increase inintracellular Ca⁺⁺ concentration that is greater than the baseline Ca⁺⁺concentration of unstimulated cells) The “% positive” value was used tomeasure the extent of activation induced by serotonin.

Actively growing NIH3T3 cells which expressed 5HT2c showed a 50% Ca²⁺response, while growth arrested cells showed a comparable 61.9% Ca⁺⁺response. Control experiments indicated that the response was receptorspecific. To elaborate, parental NIH3T3 cells which were not transfectedshowed no fluorescence change when treated with serotonin (data notshown), while pretreatment with antagonist (10 μM Mesulergine) blockedserotonin induction completely (POP Ar 10 μM 5HT, 10 μM Mes panel inFIG. 1). Only 0.1% of Mesulergine pretreated cells responded toserotonin in Ca⁺⁺ mobilization.

Example 2

These experiments were carried out to determine if the principles provenin example 1, supra, were applicable to other receptors, such as otherGq coupled receptors. To test this, cell line M1WT3 (ATCC CRL 1985) waschosen. This cell line expresses muscaranic acetylcholine receptor.Experiments were designed to determine if known agonists induce Ca²⁺mobilization in these cells after growth arrest.

M1WT3 cells were grown and treated, as set forth in example 1, supra.They were also treated with mitomycin C as described, and controls wereprepared in exactly the same way.

A Ca²⁺ imaging device was used to inspect Ca²⁺ mobilization visually. Todo this, cells were loaded with “Fura-2” a fluorescent, Ca indicator.Cells were excited at 340 and 380 nm wavelengths, and emission ratioswere monitored at 450 nm. Carbachol induced, Ca²⁺ mobilization wasobserved, in M1WT3 cells, via changes in fluorescent emission ratios,rising from approximately 0.94 to 1.03 in individual cells (FIG. 2).Mitomycin C pretreatment had no negative effect on the carbachol inducedCa²⁺ response of the cells, when compared to cells not division-arrested(data not shown). It was shown that a larger percentage of divisionarrested cells responded to carbachol (data not shown), which isconsistent with a more uniform cell population, resulting from thearrested division.

The results reported supra suggested that division arrested cells may bemore consistent, over time, in screening assays. This was tested via atime-course cell imaging experiment. Division arrested and frozen cellswere imaged as stimulated by serotonin 1, 3, 5 and 7 days followingthawing of cells. Comparable percentage, and extent of Ca response werefound, as measured by a Fura 2 fluorescence 340/380 ratio change, onthese different days, while significant changes in Ca²⁺ levels werefound in growing populations on these different days.

Example 3

It is well known that G-protein coupled receptors elicit differentpathways, depending on the G protein to which they couple. Theexperiments which follow were designed to show that seven-transmembranereceptors other than Gq coupled receptors function normally in growtharrested cells.

To do this, HEK293 cells which had been transfected stably andoverexpressed the β2 adrenergic receptor (“β2AR”) (which is a Gscoupled, seven-transmembrane receptor) was used, in experiments designedto determine if isoproterenol would induce CRE-SEAP reporter activity.

The stably transfected cells were grown in DMEM with 10% FBS, and thenwere transiently transfected with a reporter plasmid, i.e., pCRE-SEAP.The cells were treated with, mitomycin for 2 hours, 24 hours pasttransfection. The plasmid contained a CRE promoter, activity of which iselevated by cAMP, and which expresses higher levels of secreted,alkaline phosphatase (“SEAP”) upon activation of GPCRs, which use cAMPas a second messenger.

Twenty-four hours after mitomycin C treatment, β2ARs were activated with100 μM of isoproterenol, and the level of SEAP activity was measuredusing commercially available products, 24 hours later.

Actively growing β2 adrenergic receptor expressing cells responded toovernight treatment with 100 μM of isoproterenol, as measured byincreased SEAP activity (approximately 25%). Growth arrested, β2ARexpressing cells displayed much lower background SEAP activity, whichmay be attributable to mitomycin C toxicity. Notwithstanding the lowerbackground levels, overnight stimulation with isproterenol induced a 2.5fold increase in SEAP activity, thus demonstrating that growth arrestedcells still conduct largely intact signal transduction pathways down tothe transcription response, and enzyme reporter assays can be carriedout in division arrested cells.

Miyotmycin C treatment caused significant toxicity to the β2ARexpressing cells. As such, a different method for arresting celldivision was tested, i.e., gamma irradiation.

Cells were either non-irradiated, and served as a control, or wereirradiated at does ranging from 2Gy (Gray) to 8Gy. They were thentreated with 100 μM isoproterenol, as described, supra. Reporter SEAPactivity was measured, and compared to baseline activity (i.e., cellsnot treated with isoproterenol).

The results are depicted in FIG. 3. Cells which were treated with 4Gy ormore gamma irradiation showed far greater division arrest, with nonoticeable cell proliferation for about a week. These cells show normalcell morphology and, when stimulated with isoproterenol, SEAP responsesranged from 4 to 6 fold over the baseline levels. These results werecomparable to the cells that had not been division arrested, whichresponded 5.2 fold over baseline upon stimulation with isoproterenol.

The foregoing discussion sets forth features of the invention, whichrelates, inter alia, to a method for screening for a substance ofinterest. The method comprises contacting the substance of interest witha sample of division arrested cells, and determining interaction betweenthe division arrested cells and the substance of interest to determineone or more properties thereof. In this way, one can determine whether asubstance of interest has efficacy as an antagonist, an agonist, aninhibitor, a stimulator, or a modulator of cells, is toxic to the cells,and so forth.

By division arrested as used herein is meant that the cells being usedhave been treated, by means known in the art, so that either theirmitotic or meiotic cycle has been stopped, and cellular division can nolonger take place. There are many chemical, radiological, and othermethods which can be used to accomplish the arrest of cellular division,and these need not be reiterated here, as the crux of the invention isnot the act of causing the arrest of cell division, but the use of thearrested cells in assays as described.

While it is possible to treat the cells in additional ways to arrest oneor more additional biological processes, this is not necessary and,indeed, in many applications it will be desirable to have the cellsfunction normally in all other ways but for the arrest in cell division.

It will be seen by the skilled artisan that the type of cell used mayvary. Any prokaryotic or eukaryotic cell may be used, in any cell basedassay to determine the effect of a substance of interest on a cell typeof interest.

The nature of the cell type used will depend upon the particular type ofassay to be run. To this end, cells which express a particular moleculeor molecules naturally, or cells transfected or transformed to expressthe molecule or molecules of interest may be used. Prokaryotic cells,such as E. coli, which may be transformed with nucleic acid molecules,such as those which encode a eukaryotic receptor, and eukaryotic cellssuch as NIH3T3 cells, HEK293 cells, CHO cells, and so forth, can all beused. Other types of nucleic acid molecules may be used, including DNAencoding any protein of interest, RNA and antisense molecules, includingantisense DNA and antisense RNA. Many methods are known for introducingthe nucleic acid molecules to the host cells, such as via the use ofrecombinant viral vectors or other vectors that are adapted for the celltype of interest. Further, the cells may be cells which have beentransduced with a molecule of interest, such as a peptide, and/or aprotein containing a molecule such as a protein glycoprotein,lipoprotein, and so forth.

In one embodiment of the invention, the cell to be used is transformedor transfected with a nucleic acid molecule which performs a reporterfunction, such as SEAP, luciferase, green fluorescent protein, and soforth. It is well known that one of ordinary skill in the art cantransform or transfect cells with expression vectors which requireactivation of, e.g., a receptor to cause the promoter to which thereporter molecule is operably linked, to function. Since activation ofthe receptor molecule depends upon ligand receptor interaction, one candetermine the effect of a putative ligand or “anti-ligand” by measuringthe reporter molecule function, and comparing it to a control.

Of course, it will be clear to the skilled artisan that it is alsopossible to measure receptor function directly, as was shown by theexamples, supra. There are legions of receptors that are known, as istheir effect when linked to a ligand molecule. Determination of one ormore of these functions can be used as a determination of the effect ofa substance of interest.

The substance of interest may be tested directly, or it may be tested ina competitive assay, using a known antagonist or agonist of a receptoror other molecule of interest. For example, an antibody can be testedfor its efficacy as an antagonist of a molecule by mixing it with aknown ligand for the molecule, and comparing a property of the targetmolecule with and without the presence of the antibody. The converse ofthis type of assay can also be carried out, where the antibody functionis known, and the molecule of interest is not an antibody, or is in facta second antibody.

The features of this invention also afford the user a kit useful inscreening for a substance of interest. Such kits may contain, e.g., aseparate portion of each of (i) a substance which causes arresteddivision of a cell, and a substance known to interact with a targetmolecule of interest. The kit may also include cells transformed ortransfected with the molecule of interest, or cells to be transformed ortransfected and the agent used for transformation/transfection (e.g., anexpression vector), or cells naturally expressing the target molecule ofinterest or other items. All of the variations set forth supra can beused in these kits. In cases where an additional function of the cellsis to be described, that material can be included in the kit as well.

Other features of the invention will be clear to the skilled artisan,and need not be reiterated here.

1. A method for determining effect of a substance of interest on a typeof cell, comprising contacting the substance of interest to a sample ofsaid type of cell, wherein the cells in said sample have had theirdivision arrested, and determining a change in said sample of said cellsfollowing contact with said substance as a determination that saidsubstance has an effect on said type of cell.
 2. The method of claim 1,wherein said cell is a eukaryotic cell.
 3. The method of claim 1,wherein said cell is a prokaryotic cell.
 4. The method of claim 1,wherein division of said cell has been arrested during mitosis.
 5. Themethod of claim 1 wherein division of said cell has been arrested duringmeiosis.
 6. The method of claim 1, wherein said cell has beentransformed or transfected with a nucleic acid molecule which expressesa protein of interest.
 7. The method of claim 6, wherein said cell hasbeen transformed or transfected with a recombinant virus.
 8. The methodof claim 1, wherein said cell has been transformed or transfected withRNA.
 9. The method of claim 1, wherein said cell has been transformed ortransfected with an antisense nucleic acid molecule.
 10. The method ofclaim 1, wherein said cell has been transduced with a protein, peptide,or protein containing molecule.
 11. The method of claim 1, wherein saidcell is an NIH3T3 cell.
 12. The method of claim 1, said methodcomprising measuring induction of Ca²⁺ immobilization.
 13. The method ofclaim 1, wherein said cell has been transformed or transfected toproduce 5HT2c.
 14. The method of claim 1, wherein said cell expresses aG-protein coupled receptor.
 15. The method of claim 1, wherein saidcells express ion channels.
 16. The method of claim 14, wherein saidG-protein coupled receptor is muscaranic acetylcholine receptor.
 17. Themethod of claim 1, wherein said cell is an HEK293 cell.
 18. The methodof claim 1, wherein said cell expresses a .beta.2 adrenergic receptor.19. The method of claim 1, wherein said cell has been transformed ortransfected with a nucleic acid molecule which encodes a reportermolecule.
 20. The method of claim 19, wherein said nucleic acid moleculeis activated via activation of a molecule expressed in said cell. 21.The method of claim 19, wherein said reporter molecule is luciferase, orgreen fluorescent protein.
 22. The method of claim 1, said methodcomprising a competitive assay wherein said substance of interest isadmixed with a compound known to affect to said cell, and determiningany change in the known affect as a determination that said substance ofinterest affects said cell.
 23. A kit useful in screening a substance ofinterest to determine its affect on a cell type of interest, comprisinga container means which holds a separate portion of each of: (i) asubstance which arrests cell division of said cell; and, (ii) asubstance known to interact with said cell and to provoke a knownaffect.
 24. The kit of claim 23, further comprising a separate portionof (iii) a substance which affects a function other than cell divisionin said cell.